Fanconi Anemia (FA) is an autosomal recessive disease characterized by progressive bone marrow failure, leukemia susceptibility, and cellular hypersensitivity to Mitomycin C and other DNA crosslinkers. The thirteen known FA proteins cooperate in a common DNA repair pathway, the FA pathway (D'Andrea, A.D. Susceptibility pathways in Fanconi's anemia and breast cancer. N Engl J Med 2010;362:1909-19). A critical downstream event in the FA pathway is the monubiquitination of the FANCD2/FANCI heterodimer. This heterodimer is subsequently deubiquitinated by a multisubunit DUB enzyme complex, USP1/UAF1. During the last five-year grant period, we have shown that the USP1/UAF1 complex plays a critical regulatory function in the FA pathway. The work has had high impact in several research fields. First, understanding the molecular pathology of FA has elucidated several features of genomic instability, leukemia, and bone marrow failure in FA patients and in the general (non-FA) human population. Second, the FA pathway has provided insight into the mechanism of DNA crosslink repair. Third, the FA pathway has provided novel insights to the general field of ubiquitin biology. The FA pathway has uncovered several regulatory mechanisms which control the monoubiquitination state of a critical protein, FANCD2-Ub. This new application describes a novel, and perhaps the most highly regulated, step in the FA pathway - namely, the timed deubiquitination of the FANCD2-Ub substrate by USP1/UAF1 after DNA damage. The finding provides important general insights to the mechanism of DUB/substrate recognition. During the next five year study period our specific aims are: 1) To test the hypothesis that the USP1/UAF1 deubiquitinating complex is targeted to its FANCD2/FANCI substrate through a SLD (Sumo-Like Domain)/SIM interaction. 2) To examine the functional interaction between FANCD2/FANCI and PCNA. 3) To further characterize mouse knockout models of USP1 and UAF1 and to identify other USP1/UAF1 substrates. PUBLIC HEALTH RELEVANCE: Fanconi Anemia (FA) is a rare recessive genetic disease causing bone marrow failure and a predisposition to cancer. Through the systematic study of this disease over the last fifteen years, my laboratory has contributed significantly to the elucidation of a biochemical pathway in human cells regulating DNA repair. The Fanconi Anemia proteins function in this pathway to regulate normal blood cell formation and to prevent the onset of cancer;disruption of this pathway leads to anemia and cancer. Now that we have established this pathway, the protein biomarkers we have described will be useful in 1) diagnosing anemia and cancer in the general population, and 2) developing drugs to treat anemia and cancer in the general population. Thus, the study of this disease will have a broad impact on public health.